Hotstart taq polymerase

Oligonucleotide primers used for the mycobacterial hsp65 gene were Tb11 (5′-CAACGATGGTGTGTCCCAT-3′) and Tb12 (5′-CTTGTCGAACCGCATACCCT-3′). Briefly, the PCR reactions contained 3 μL of 10X buffer, 1.8 μL of 15 mM MgCl2, 3 μL of Q solution, 0.6 μL of 10 mM dNTP mix, 1.8 μL of each primer, 0.2 μL of hot-start Taq polymerase from Qiagen, 14.8 μL of nuclease-free water, and 3 μL of template DNA. Cycling conditions were (a) initial denaturation at 95°C for 15 min and 35 cycles of denaturation at 96°C for 1 min, (b) annealing at 60°C for 1 min, (c) extension at 68°C for 1 min, and (d) final extension at 72°C for 10 min. Amplification of the 441-bp product was confirmed by 1.5% agarose gel electrophoresis and UV transillumination. PCR products were purified and sequenced by outsourcing (Macrogen Europe). Vector sequences in the sequenced hsp65 genes were removed, the sequences were processed into gap experimental files with pregap4, edited with gap4 of the Staden package,[20 (link)] and the corresponding consensus sequence was saved in FASTA format for further analysis. The consensus hsp65 gene sequence of each isolate was analyzed by NCBI nucleotide blast against the microbial database of representative genomes only and selecting highly similar sequences (megablast), whereas leaving all other settings to default.